P167	Shear stress induced FGF-2 release from endothelial cells is mediated by elastolytic activity.
1Chr.Klarskov, 2F.Krötz, 1U.Pohl, 1T.Gloe
1Institute of Physiology, LMU, München, DE; 2Klinikum Innenstadt, LMU, München, DE.

Basic fibroblast growth factor (FGF-2) a cytokine stored preformed in substantial amounts within endothelial cells plays a pivotal role in vascular remodelling by regulating migration, proliferation, and new vessel formation. It release mechanism however is not understood completely since it is lacking a classical secretion sequence necessary for vesicle-transport. Recently, we could show that shear stress induced release of bFGF is mediated by the integrin αvβ3 (JBC, 277:23453). Based on these findings we investigated whether matrix modulations by proteases contribute to signalling cascades leading to enhanced bFGF liberation from endothelial cells. Porcine aortic endothelial cells subjected to shear stress (16 dyn/cm2) exhibited a significant higher elastase-like activity in their supernatant media from 5 to 15 mU/ml (p<0.05). At the same time those cells released a 10-fold higher FGF-2 amount into conditioned media than their static controls (p<0.05, n=6). Presuming that the enhanced elastolytic activity is essential for the FGF-2 release, a serine protease inhibitor (aprotinin, 1µg/ml) was added to the cells before the onset of shear stress. Indeed, inhibition of elastase significantly reduced the shear stress induced FGF-2 liberation. Moreover, even static cells treated with elastase alone (0.5 U/ml) showed an increased FGF-2 cell surface staining (FACS and histochemistry) which went along with a 1.8-fold higher FGF-2 release compared to control cells (p<0.05, n=6). As for shear stress, this protease-induced release could be prevented by inhibition of integrin αvβ3. Further results indicate that Hsp27 was phosphorylated upon elastase treatment, which could be prevented by inhibition of integrin αvβ3. The phosphorylation of Hsp27 was shown to be dependent on the p38 MAP Kinase pathway and phospho-Hsp27 co-precipitated with FGF-2 verifying previous findings, suggesting a function of Hsp27 in FGF-2 release. The results presented here indicate that mechano-sensing of shear stress induces the release of FGF-2. This release mechanism is critically dependent on proteolytic matrix modulation via elastase and is subsequently regulated by specific cell-matrix interactions via the integrin αvβ3. Moreover, Hsp27 phosphorylation may play a central role in the involved intracellular signalling cascade and transport machinery.

Copyright © 2005 S. Karger AG, Basel. Any further use of this abstract requires written permission from the publisher.